Novel DNA-binding specificity can be engineered into Cys2His2 zinc finger proteins by either selection or design, and they can be used to deliver a DNA cleavage domain to a specific address in a genome. These tailor-made restriction endonucleases (Zinc Finger Nucleases or ZFNs), which function as (hetero)dimers, can introduce double stranded breaks at a specific genomic location to inactivate a target gene through imprecise repair or recode a target gene through homologous recombination with an exogenously supplied donor DNA. ZFN technology can potentially be used to apply simple or sophisticated reverse genetic approaches to a broad range of metazoan systems that were previously only accessible in the mouse and fly. This technology, which should also find application in bioengineering and human gene therapy, is still in its infancy. The current generation of ZFNs has not been thoroughly characterized nor completely optimized. We propose to develop a new generation of artificial nucleases with improved properties (activity, precision, range of targetable sequences) that will fully realize the potential of this technology for tailored genome editing. In Aims 1 & 2 we will define the optimal assembly of ZFPs and the optimal fusion of the nuclease domain to create heterodimeric ZFNs that are both efficient and precise. We will also develop new nuclease architectures to expand the types of DNA sequences that can be targeted. In Aim 3 we will explore the potential of ZFNs to perform knockouts of highly related gene families, which would allow complex knockout combinations to be rapidly created.